Purpose :
There is an increasing number of human gene therapy and stem cell surgery trials that require subretinal injection to achieve therapeutic effect. However, there is scant data to support the surgical techniques and instrumentation used in these studies. Moreover, it is unknown whether higher injection rates can cause retinal damage; it is also unclear whether blebs from larger injection cannulas can still result in retinal reattachment without air-fluid exchange. We sought to analyze outcomes from several key components of surgery including: 1) retinal reattachment with variably sized needle-cannulas and 2) effect of injection rate on retinal morphology.

Methods :
Yucatan mini pigs underwent vitrectomy and subretinal injection (by a surgical assistant) of 300uL of viral vector, iPS-derived RPE/photoreceptor precursor cells, or control buffers using a 31G or 41G polyamide cannula. Air-fluid exchange was not done. Indirect ophthalmology was performed at sacrifice (POW1-POM3) to detect retinal reattachment and RPE changes. Morphology of eyes was assessed on H&E stained paraffin sections using light microscopy. Immunofluorescence staining with anti-RPE65 antibody was used to detect RPE changes in a subset of eyes with pseudo-GA. Additionally, cadaveric pig eyes underwent vitrectomy and subretinal injection of 300uL of buffer at 1.8mL/min or 0.18mL/min using an automated injector. Eyes were immediately fixed in 4% paraformaldehyde then embedded in paraffin for morphologic analysis.

Results :
For the survival surgeries, 100% had spontaneous retinal reattachment using both 31G (n=32 eyes) and 41G (n=32) cannulas. RPE changes or pseudo-GA was seen in 12/32 eyes with the 41G cannula and 22/32 eyes with the 31G cannula. Histologically, of 11 eyes with pseudo-GA seen on exam, RPE was relatively intact but there was depigmentation of the apical villi in 6 eyes; loss of RPE65 expression was seen in 8. Of cadaveric eyes injected at a speed of 1.8mL/min, there was significant loss of RPE cells in 5/6 eyes compared to 0/2 injected at 0.18mL/min (p<0.05).

Conclusions :
Both 31G and 41G cannulas can be used to successfully administer subretinal injections that will spontaneously reattach without need for air fluid exchange in pigs. Higher bleb injection speeds may result in RPE changes or pseudo-GA seen post-operatively on ophthalmoscopy that correlates to RPE depigmentation and loss of RPE65 expression.

This is an abstract that was submitted for the 2016 ARVO Annual Meeting, held in Seattle, Wash., May 1-5, 2016.